The search for new therapeutic agents has been greatly aided in recent years by a better understanding of the structure of enzymes and other biomolecules associated with diseases. One important class of enzymes that has been the subject of extensive study is protein kinases.
Protein kinases constitute a large family of structurally related enzymes that are responsible for the control of a variety of signal transduction processes within the cell. (See, Hardie, G. and Hanks, S. The Protein Kinase Facts Book, I and II, Academic Press, San Diego, Calif.: 1995). Protein kinases are thought to have evolved from a common ancestral gene due to the conservation of their structure and catalytic function. Almost all kinases contain a similar 250-300 amino acid catalytic domain. The kinases may be categorized into families by the substrates they phosphorylate (e.g., protein-tyrosine, protein-serine/threonine, lipids, etc.). Sequence motifs have been identified that generally correspond to each of these kinase families (See, for example, Hanks, S. K., Hunter, T., FASEB J., 9:576-596 (1995); Knighton et al., Science, 253:407-414 (1991); Hiles et al., Cell, 70:419-429 (1992); Kunz et al., Cell, 73:585-596 (1993); Garcia-Bustos et al., EMBO J., 13:2352-2361 (1994)).
In general, protein kinases mediate intracellular signaling by effecting a phosphoryl transfer from a nucleoside triphosphate to a protein acceptor that is involved in a signaling pathway. These phosphorylation events act as molecular on/off switches that can modulate or regulate the target protein biological function. These phosphorylation events are ultimately triggered in response to a variety of extracellular and other stimuli. Examples of such stimuli include environmental and chemical stress signals (e.g., osmotic shock, heat shock, ultraviolet radiation, bacterial endotoxin, and H2O2), cytokines (e.g., interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α)), and growth factors (e.g., granulocyte macrophage-colony-stimulating factor (GM-CSF), and fibroblast growth factor (FGF)). An extracellular stimulus may affect one or more cellular responses related to cell growth, migration, differentiation, secretion of hormones, activation of transcription factors, muscle contraction, glucose metabolism, control of protein synthesis, and regulation of the cell cycle.
Many diseases are associated with abnormal cellular responses triggered by protein kinase-mediated events as described above. These diseases include, but are not limited to, autoimmune diseases, inflammatory diseases, bone diseases, metabolic diseases, neurological and neurodegenerative diseases, cancer, cardiovascular diseases, allergies and asthma, Alzheimer's disease, and hormone-related diseases. Accordingly, there has been a substantial effort in medicinal chemistry to find protein kinase inhibitors that are effective as therapeutic agents.
Among medically important serine/threonine kinases is the family of polo-like kinases that have an important role in several stages of mitosis. This family includes the mammalian PLK1, PLK2 (serum-inducible kinase, Snk), PLK3 (fibroblast growth factor-inducible kinase, Fnk/proliferation-related kinase, Prk) and PLK4 (Sak). Polo-like kinase 1 (PLK1) has been suggested to promote mitotic entry by activating cyclin B/cdk1 by phosphorylating cyclin B itself, by phosphorylating the Cdk1-activating phosphatase Cdc25c and by phosphorylating the Cdk1-inhibiting kinases Myt1/Wee1 (Abrieu, A., et al. Cell Sci 111, 1751-1757 (1998); Jackman, M. et al Nat Cell Biol. 5, 143-148 (2003); Nakajima, H. et al. J. Biol. Chem. 278, 25277-25280 (2003); Qian, Y. W. et al. Mol. Cell. Biol. 18, 4262-4271 (1998); Toyoshima-Morimoto, F. et al. Nature 410, 215-220 (2001); Watanabe, N. et al. Proc. Nat. Acad. Sci. USA 101, 4419-4424 (2004)). Furthermore, PLK1 has been implicated in centrosome maturation and separation, with defects giving rise to monopolar spindles (Lane, H. A., et al. J. Cell Biol. 135, 1701-1713 (1996); Sunkel, C. E., et al. J. Cell Sci. 89, 25-38 (1988)). Studies on human cells and in vitro have indicated a role for PLK1 in activating the anaphase-promoting complex/cyclosome (APC/C) (Golan, A. et al J. Biol. Chem. 277, 15552-15557 (2002); Kotani, S. et al. Mol. Cell 1, 371-380 (1998). PLK1 is located mostly in the cytoplasm during interphase and translocates to the nucleus in early mitosis. PLK1 also has been shown to associate with centrosomes from G2 up to metaphase, to translocate to kinetochores at metaphase, and to locate at the midbody from anaphase to telophase (Golsteyn, R. M. et al. J. Cell Biol. 129, 1617-1628 (1995); Lee et al. Mol. Cell Biol. 15, 7143-7151 (1995)).
In contrast to PLK1, PLK2 and PLK3 appear to perform different functions, and PLK3 plays some functions that might directly antagonize PLK1 function (Smits et al. Nat. Cell Biol. 2 672-676 (2000); Xie et al. J. Biol. Chem. 276, 43305-43312 (2001). Small interfering RNA (siRNA)-mediated inhibition of PLK3 expression suggests that PLK3 is essential for superoxide-induced cell death, and deletion analysis of PLK3 showed that the N-terminal domain (amino-acids 1-26) is essential for induction of delayed onset of apoptosis (Li Z. et al. J. Biol. Chem. 280, 16843-16850 (2005)). Over-expression of PLK1 has been observed to cause oncogenic transformation in NIH 3T3 cells (Smith et al. Biochem. Biophys. Res. Commun. 234, 397-405 (1997), while overexpression of PLK3 induces apoptosis (Conn et al. Cancer Res. 60, 6826-6831 (2000).
Polo-like kinases are characterized by an N-terminal catalytic Ser/Thr kinase domain and a C-terminal non-catalytic region containing two tandem Polo-boxes (Sonnhammer et al. Nucleic Acids Res. 26, 320-322 (1998). The Polo-box domain is required for correct subcellular localization (Lee et al., Proc. Natl. Acad. Sci. USA 95, 9301-9306 (1998); Ma et al., Mol. Cancer. Res. 1, 376-384 (2003); May et al., J. Cell Biol. 156, 23-28 (2002); Reynolds & Ohkura J. Cell Sci., 116, 1377-1387 (2003)), and the crystal structure of the polo-box domain in PLK1 has been determined (Elia et al. Cell 115, 83-95 (2003); Cheng et al., EMBO J. 22, 5757-5768 (2003)). The Polo-box domain recognizes a conserved phosphothreonine/serine motif in the substrate which binds along a positively-charged cleft located at the edge of the Polo-box interface. Mutations of key residues to disrupt the phosphodependent interactions abolishes cell-cycle dependent localization but does not affect its kinase activity (Lee et al., Proc. Natl. Acad. Sci. USA 95, 9301-9306 (1998), and suggests that substrate binding to the Polo-box domain is required for proper mitotic progression (Elia et al. Cell 115, 83-95 (2003)).
Because of their link to cellular proliferation, PLK family proteins have been associated with cancer development and progression. In fact, the over-expression of murine PLK1 in NIH3T3 cells has been observed to be transforming (Smith, M. R. et al. Biochem. Biophys. Res. Commun. 234, 397-405 (1997)). In addition, PLK1 has been seen to be over-expressed in a wide variety of primary tumor cell lines, including breast cancer (Wolf G. et al. Pathol. Res. Pract. 196, 753-759 (2000)), colorectal cancer (Takahashi T. et al. Cancer Sci. 94, 148-152 (2003)), non-small-cell lung cells (Wolf G. et al. Oncogene 14, 543-549 (1997)), head and neck squamous cell carcinomas (Knecht R. et al. Cancer Res. 59, 479-480 (1999)), ovarian carcinomas (Takai N. et al. Cancer Lett. 164, 41-49 (2001)), prostate (Weichert W. et al Prostate 60, 240-245 (2004)) and pancreatic cancer (Gray P. J. et al. Mol. Cancer. Ther. 3, 641-646 (2004)) and is positively correlated with aggressiveness and prognosis (Takai N., et al Oncogene 24, 287-291 (2005)).
Whilst the precise function of PLK2 is uncertain, the expression of PLK3 is negatively correlated with the development of certain cancers. PLK3 mRNA is either undetectable or markedly downregulated in lung carcinomas (Li B. et al. J. Biol. Chem. 271, 19402-19408 (1996)) head and neck squamous cell carcinomas (Dai W. et al. Genes Chromosomes Cancer 27, 332-336 (2000)) and in colon tumors (Dai W. et al. Int. J. Oncol. 20, 121-126 (2002)). In support of these observations, the expression of PLK3 in vitro reduces the rate of fibroblast cell proliferation (Dai W. et al. Genes Chromosomes Cancer 27, 332-336 (2000)) and the enforced expression of constructs expressing kinase-active PLK3 induces rapid cell cycle arrest and apoptosis (Conn C. W. et al. Cancer Res. 60, 6826-6831 (2000); Wang Q. et al. Mol. Cell. Biol. 22, 3450-3459 (2002)). PLK3 has been shown to be a stress response protein involved in DNA damage checkpoint (Bahassi E. M. et al. Oncogene 21, 6633-6640 (2002); Xie S. et al. J. Biol. Chem. 276, 43305-43312 (2001)). In this role, PLK3 only becomes phosphorylated following DNA damage or mitotic spindle disruption, whereupon its kinase activity is enhanced, otherwise, PLK3 is not phosphorylated during normal cell cycle progression. Consistent with this, possible substrates of PLK3 are thought to include both the tumor suppressors Chk2 and p53, which have both been shown to bind to PLK3 in vivo, and be phosphorylated by PLK3 in vitro.
The role of PLK3 in DNA damage checkpoint leads to the interest in designing PLK3 inhibitors that are effective as therapeutic agents. A challenge has been to find protein kinase inhibitors that act in a selective manner, targeting only PLK3. Since there are numerous protein kinases that are involved in a variety of cellular responses, non-selective inhibitors may lead to unwanted side effects. In this regard, the three-dimensional structure of the kinase would assist in the rational design of inhibitors. The determination of the amino acid residues in PLK3 binding pockets and the determination of the shape of those binding pockets would allow one to design selective inhibitors that bind favorably to this class of enzymes. The determination of the amino acid residues in PLK3 binding pockets and the determination of the shape of those binding pockets would also allow one to design inhibitors that can bind to PLK3.
For example, a general approach to designing inhibitors that are selective for an enzyme target is to determine how a putative inhibitor interacts with the three dimensional structure of the enzyme. For this reason it is useful to obtain the enzyme protein in crystal form and perform X-ray diffraction techniques to determine its three dimensional structure coordinates. If the enzyme is crystallized as a complex with a ligand, one can interactively elucidate the binding pocket, and determine both the shape of the enzyme binding pocket when bound to the ligand, as well as the amino acid residues that are capable of close contact with the ligand. By knowing the shape and amino acid residues in the binding pocket, one may design new ligands that will interact favorably with the enzyme. With such structural information, available computational methods may be used to predict how strong the ligand binding interaction will be. Such methods thus enable the design of inhibitors that bind strongly, as well as selectively to the target enzyme.
Despite the fact that the genes for PLK3 has been isolated and the amino acid sequence of PLK3 is known, no one has described X-ray crystal structural coordinate information of PLK3 protein. As discussed above, such information would be extremely useful in identifying and designing potential inhibitors of the PLK3 kinase or homologues thereof, which, in turn, could have therapeutic utility.
The structures of several serine/threonine kinases have been solved by X-ray diffraction and analyzed. Specifically, the crystal structures of P38 kinase (Wilson et al., J. Biol. Chem., 271, pp. 27696-27700 (1996)) and MAPKAP Kinase 2 (U.S. Provisional application 60/337,513) have been studied in detail.
To date, no crystal structures of PLK3 kinase have been reported. Thus the crystal structure of unphosphorylated PLK3 kinase domain complexes with inhibitors are of great importance for defining the active conformation of PLK3 kinase. This information is essential for the rational design of selective and potent inhibitors of PLK3.